Protozoan parasites
(Cryptosporidium, Giardia, Cyclospora)
With C.R. Fricker1 and H.V. Smith2
2
3
Water Quality Centre, Thames Water Utilities
Reading, United Kingdom
Scottish Parasite Diagnostic Laboratory, Stobhill Hospital, NHS Trust
Glasgow, United Kingdom
Chapter 1
The authors wish to thank Morteza Abbaszadegan, Jamie Bartram, Phil Berger,
Joe Cotruvo, David Cunliffe, dr. Feuerpfeil, Arie Havelaar, Henk Ketelaars, Mark
LeChevallier, Yasumoto Magara, Ynez Ortega, E. Pozio, Stig Regli, Joan Rose,
Steven Schaub, Ciska Schets, Susan Shaw and all the participants of the
meeting on the Rolling Revision of the WHO Guidelines for Drinking-water
quality in Medmenham, 17 to 21 March 1998 for critical reading of the
manuscript. Their useful comments have improved the comprehensiveness of
the description of the current knowledge of these parasites and of the means to
control waterborne transmission.
This paper will be published as addendum to the Guidelines for Drinking-water
Quality of the World Health Organisation, Geneva, Switzerland.
10
Protozoan parasites
WATERBORNE PROTOZOAN PARASITES
Several species of parasitic protozoa are transmitted through water. Of these,
Giardia intestinalis and Entamoeba histolytica/dispar are long recognised as the
most common intestinal parasites throughout the world. Morbidity and, in
particular for E.histolytica/dispar, mortality rates are high, especially in nonindustrialised countries. More information on Entamoeba can be found in volume
2 of the WHO Guidelines for Drinking Water Quality and in the informal
consultation document on enteric protozoa (1994).
A wide variety of free-living amoebae is capable of multiplication in (drinking)
water, but only few species have been identified as pathogenic for man. These
are Naegleria fowleri and Acanthamoeba spp. Naegleria fowleri can be present
in thermally polluted waters and sporadically causes lethal primary amoebic
meningoencephalitis. Only one outbreak has been related to a drinking water
supply system (Marshall et al.,1997). Acanthamoeba can be found in the entire
aquatic environment. It sporadically causes keratitis in contact lens wearers
after exposure to contaminated recreational water and contact lens cleaning
fluids (Marshall et al., 1997). Drinking water taps were identified as the source
of contamination of home-made lens cleaning solutions containing
Acanthamoeba (Seal et al., 1995). Acanthamoeba has also been suggested as a
vehicle for environmental transmission of Legionella bacteria (Campbell et al.,
1995).
The increasing population of severely immunocompromised people, due to the
AIDS epidemic, cancer chemotherapy and organ transplants, has increased the
prevalence of opportunistic infections and has led to the recognition of the
disease causing potential of other intestinal protozoan parasites, such as
Cryptosporidium parvum, Cyclospora sp. and Microsporidia as human
pathogens.
The first human cases of cryptosporidiosis were reported in 1976 (Meisel et al.,
1976; Nime et al., 1976) and Cryptosporidium was first thought to be an
opportunistic pathogen of immunocompromised persons. The recognition of
frequent cases in immunocompetent individuals and a number of waterborne
outbreaks have changed this image. C. parvum is now recognised as one of the
most commonly identified intestinal pathogens throughout the world. It’s
relative occurrence is dependent on factors such as age and other demographic
characteristics of the study population and season. In children at the age of 1-5
with diarrhoea, it can be the most frequently found pathogen (Palmer, 1990).
Cyclospora sp. has recently been recognised as a waterborne pathogen. Initially,
it was referred to as cyanobacterium-like bodies but is now classified as
Cyclospora sp. (Bendall et al., 1993; Ortega et al., 1993). It has been
associated with several waterborne outbreaks world-wide.
Microsporidia are a large group (almost 1000) of species and are widely
distributed in nature (Casemore, 1996). Although they were recognised as
pathogens in fish, birds and some mammals, several species have recently been
identified as cause of disease in severely immunocompromised humans. These
species have primarily been associated with infections of the intestinal tract, but
11
Chapter 1
dissemination to the biliary, urinary and respiratory tract may occur. Some
species have been implicated in ocular infections in immunocompetent persons.
The mode of transmission is still unclear, but a faecal-oral route is likely.
Waterborne transmission has not been demonstrated, but their persistence in
water, resistance to disinfection and small size (some as small as 1-2 µm)
suggest that this must be considered possible, especially for
immunocompromised individuals.
Toxoplasma gondii is a coccidian parasite that has long been recognised as
human pathogen. It is an intracellular parasite with felines as the definitive host.
These are infected primarily by the consumption of infected mammals and birds,
that act as secondary hosts. In these secondary hosts, the parasite settles itself
as tissue cysts in muscle and brain tissue. Only felines carry the parasite in their
intestinal tract and shed oocysts that sporulate in the environment. The oocysts
are 10-12 µm and can survive in water and moist soils for long periods of time.
Consumption of undercooked meats and raw milk and contact with feline faeces
(cat litter, sand boxes) are the primary sources of Toxoplasma infections in
humans (Guy, 1996). Two waterborne outbreaks have been reported. Both were
believed to have been derived from contamination of water by cat faeces.
This review focuses on Cryptosporidium parvum, Giardia intestinalis and
Cyclospora sp., since these are the parasites of primary concern to drinking
water supply and a large amount of information on waterborne transmission has
accumulated from recent research.
Significance of Cryptosporidium and Giardia as waterborne pathogens
Oocysts of Cryptosporidium and cysts of Giardia are ubiquitously present in the
aquatic environment. They have been found in most surface waters, their
concentration being related to the level of faecal pollution or human use of the
surface water (Hansen & Ongerth, 1991; LeChevallier et al., 1991).The
environmentally robust (oo)cysts are very persistent in water (DeReignier et al.,
1989; Robertson et al., 1992; Chauret et al., 1995) and are extremely resistant
to the disinfectants commonly used in drinking water treatment (Hibler et al.,
1987; Korich et al., 1990; Finch et al., 1993a.b). These characteristics, coupled
with the low numbers of (oo)cysts required for an infection (Rendtorff, 1954;
Dupont et al., 1995; Okhuysen et al., 1998) make these organisms the most
critical pathogens for the production of safe drinking water from surface water
with disinfection and for post-treatment contamination. Well protected
groundwaters, that are not under the influence of surface water or other
sources of contamination, are free of these (and other) enteropathogens. If
abstraction, treatment and distribution of these waters are properly designed
and operated, the risk of faecal contamination is very low and they will not be a
source of waterborne transmission of parasitic protozoa. Groundwaters that are
under the influence of surface water or other contamination sources (surface
run-off) can be contaminated with, low levels of, Cryptosporidium and Giardia
(Hancock et al., 1997) and cause waterborne illness (Craun et al., 1998).
Treatment of these waters with disinfection alone offers no protection against
12
Protozoan parasites
Cryptosporidium and only limited protection against Giardia. Hence, filtration of
these waters is necessary to produce safe drinking water.
Many waterborne outbreaks of giardiasis and cryptosporidiosis have been
reported in industrialised countries (Craun, 1990; MacKenzie et al., 1994; Craun
et al., 1998). In these outbreaks, (oo)cysts have entered the drinking water
because of surface water treatment failure, (increased) contamination of the
source water and leakage into the distribution system.
In a significant number of these outbreaks, the drinking water that was
implicated as the cause of the outbreak complied with the WHO-guidelines for
Escherichia coli levels and turbidity (Craun 1990; Craun et al., 1998). In most
outbreaks, deviations from normal raw water quality or treatment operation
could be identified. However, in a drinking waterborne outbreak in Las Vegas,
no abnormalities in operation or water quality (raw or treated) were detected
(Goldstein et al., 1996).
The occurrence of outbreaks in the absence of a warning signal from the routine
water quality monitoring for coliforms that the water may be contaminated is a
severe shortcoming of the coliforms as indicator for microbiologically safe
drinking water, which calls for additional means to safeguard drinking water.
THE PARASITES AND THE DISEASE
Cryptosporidium parvum
Taxonomy
Members of the genus Cryptosporidium (Apicomplexa, Cryptosporidiidae) are
small coccidian protozoan parasites that infect the microvillous region of
epithelial cells in the digestive and respiratory tract of vertebrates. Several
species of Cryptosporidium have been described. These species appear to be
specific for a class of vertebrates: C. parvum, C. muris, C. felis and C. wrairi
infect mammals, C. baileyi and C. meleagridis infect birds, C. serpentis infects
reptiles and C. nasorum tropical fish. Infections in humans are almost
exclusively caused by C. parvum. This species is also frequently found in
infections of cattle and sheep and causes infections in a wide range of other
mammal species.
Life-cycle
Infected hosts shed oocysts, the environmentally resistant transmission stage of
the parasite, with their faeces (Fayer & Ungar,1986, Fayer et al., 1997). These
oocysts are immediately infectious and may remain in the environment for very
long periods without losing their infectivity, due to a very robust oocyst wall
that protects the four sporozoites against physical and chemical damage. When
the oocyst is ingested by a new host, the suture in the oocyst wall opens
(excystation), triggered by the body temperature and the interaction with
stomach acid and bile salts. Four motile sporozoites are released that infect the
epithelial cells of the small intestine, mainly in the jejunum and ileum. The
parasite infects the apex of the epithelial cells and resides beneath the cell
13
Chapter 1
membrane of the epithelial cells but outside of the cytoplasm. The sporozoites
transform into several life stages in an asexual (merogony) and a sexual
reproduction cycle (gametogony). The oocysts are the result of the sexual
reproduction cycle. Oocysts of C. parvum are spherical with a diameter of 4-6
µm. Thick- and thin-walled oocysts are formed. The thin-walled oocysts may
excyst within the same host and start a new life cycle (autoinfection). This may
lead to a heavily infected epithelium of the small intestine, resulting in
malabsorptive or secretory diarrhoea. The thick-walled oocyst is excreted with
the faeces and is environmentally robust.
Pathogenicity
Infection studies with healthy human volunteers show a very good relation
between probability of infection and the ingested oocyst dose of a bovine C.
parvum strain (Dupont et al., 1995). At the lowest dose (30 oocysts) the
probability of infection was 20%. This probability increased to 100% at 1000
oocysts. When the dose-response data are fitted with an exponential model, the
probability of infection (Pi) is described by:
Pi = 1-e-r x dose , where r, the dose-response parameter, is 0.004005 (95% CI
0.00205 - 0.00723) for this C. parvum strain (Teunis et al., 1996). This
approach assumes that ingestion of even a single oocyst results in a distinct
probability of infection (of 0.5%). Although there was a clear dose-response
relation for infection, occurrence of symptoms of intestinal illness in the
volunteers was not dose related. Recent studies indicate that the relation
between oocyst dose and probability of infection and illness varies between C.
parvum strains (Chappell, pers. comm)
The disease
The average incubation period is around 7 days, but shows a strong variation
(Ungar, 1990; Dupont et al., 1995). Watery diarrhoea is the most prominent
symptom of an intestinal infection with C. parvum (Fayer & Ungar, 1986;
Ungar, 1990). Frequent and voluminous bowel movements can cause
dehydration and weight loss (Arrowood, 1997). Other symptoms are nausea,
abdominal cramps, vomiting and mild fever. MacKenzie et al. (1994) compared
clinical data from cases detected by (passive) laboratory surveillance with cases
detected by (active) telephone surveys during the 1993 Milwaukee waterborne
outbreak, which involved 400 000 patients. Patients who submitted a stool
sample for laboratory diagnosis suffered more serious disease, as manifest from
the higher prevalence of the following complaints in these patients: fatigue, loss
of appetite, nausea, fever, chills and sweats, and vomiting.
In immunocompetent persons, the infection is limited by the immune response
that eventually clears the host of the parasite. The occurrence of persistent and
heavy infections in patients with deficiencies in the cellular (AIDS,
chemotherapy, congenital) or humoral (congenital hypogammaglobulinaemia)
immune responses suggests that both types of immune response are needed to
limit and clear the infection. Several animal studies suggest that the immune
response results in protection against re-infection (Zu et al., 1992). Protective
immunity in humans is suggested by the high rates of asymptomatic carriage in
14
Protozoan parasites
countries with a high prevalence of cryptosporidiosis. Also, infected volunteers
that were rechallenged with the same strain one year after the initial infection
were significantly less sensitive to (re)infection (Okhuysen et al., 1998).
However, the rates of diarrhoea were similar in both exposures, but the illness
was less intense in the re-infected volunteers, which indicates some degree of
protective immunity.
The duration of the infection is generally 7-14 days for the immunocompetent,
but also 23-32 days have been reported as median duration of the infection (van
Asperen et al., 1996). The peak intensity of oocysts shedding, with an average
concentration of 106/g, coincides with the peak intensity of clinical symptoms.
Oocyst shedding lasts for at least 2 weeks in 82% of the infected persons, 42%
shed oocysts for at least 3 weeks and 21% for at least 4 weeks (Baxby et al.,
1985). Again, there is a difference between cases from laboratory surveillance
(duration 2-4 weeks) and cases in the general population (duration typically 3-6
days). Relapses of diarrhoea are commonly seen, both population based
(outbreak) studies and in volunteer experiments report 1-5 additional episodes in
40-70% of patients. This phenomenon considerably increases the mean duration
of disease and its variability.
The mortality in immunocompetent patients is generally low. In immunodeficient
persons however, the infection can be persistent and severe (Ungar, 1990)
resulting in very profuse diarrhoea that leads to severe dehydration. Severe
infections have been reported in patients with concurrent infections (AIDS, but
also measles, chicken pox), persons with congenital immune deficiencies,
persons receiving immunosuppresive drugs (for cancer therapy, transplants or
skin lesions) and malnourished persons (Fayer et al., 1997). Also, pregnancy
may predispose to Cryptosporidium infection (Ungar, 1990). The prevalence of
cryptosporidiosis in AIDS patients in industrialised countries is around 10-20%
(Current & Garcia, 1991). In the absence of an effective immune response, the
infection may spread throughout the entire intestinal tract and to other parts of
the body (gall bladder, pancreas, respiratory tract). Despite extensive effort, no
consistently effective therapeutic agent has been found (Blagburn & Soave,
1997). Immunotherapy with monoclonal antibodies or hyperimmune bovine
colostrum have been reported to resolve diarrhoea in AIDS patients at least
temporarily (Riggs, 1997). Similar findings were reported for several
chemotherapeutic agents (azithromycin, paromomycin) (Blagburn & Soave,
1997).
The severe dehydration, the spread of the infection and the lack of an effective
therapy lead to a high mortality in immunodeficient patients. Accurate data are
lacking. In one study in the UK, 19% of the AIDS patients with cryptosporidiosis
were suspected to have died from the infection (Connolly et al., 1988). A
compilation of case reports of cryptosporidiosis resulted in a mortality rate of
46% in AIDS patients and 29% in patients with other immunodeficiencies (Fayer
& Ungar, 1986).
Prevalence
15
Chapter 1
In stool surveys of patients with gastro-enteritis, the reported prevalence of
Cryptosporidium is 1-4% in Europe and North America and 3-20% in Africa,
Asia, Australia, South and Central America (Current & Garcia, 1991). Peaks in
the prevalence in developed countries are observed in the late summer (van
Asperen et al., 1996) and in spring (Casemore, 1990).
Asymptomatic carriage, as determined by stool surveys, generally occurs at
very low rates in industrialised countries (<1%) (Current & Garcia, 1991),
although in day care centres higher rates have been reported (Lacroix et al.,
1987; Crawford & Vermund, 1988; Garcia-Rodriguez et al., 1989). Routine bile
endoscopy suggests a higher asymptomatic prevalence: 13% of non-diarrhoeic
patients were shown to carry Cryptosporidium oocysts (Roberts et al., 1989).
High rates of asymptomatic carriage (10-30%) are common in non-industrialised
countries (Current & Garcia, 1991). Seroprevalence rates are generally higher
than faecal carriage rates, from 25-35% in industrialised countries up to 95% in
South America (Casemore et al, 1997). Seroprevalence rates increase with
increasing age (Zu et al., 1992; Kuhls et al., 1994) and are relatively high in
dairy farmers (Lengerich et al., 1993) and day care centre attendants (Kuhls et
al., 1994).
Routes of transmission
A major route of exposure is person-to-person transmission, as illustrated by
outbreaks in day-care centres (Fayer & Ungar, 1986; Casemore, 1990; Cordell
& Addiss, 1994) and the spread of these outbreaks in the households of the
attending children. Also sexual practices that imply oro-anal contact yield a high
risk for exposure to Cryptosporidium. Transmission from animals (mammals) to
man occurs, especially from newborn animals. Many infections have been
derived from contact with infected calves and lambs (Casemore, 1990). Also
pet animals can be infected with oocysts, but appear to be no important source
of human infection (Casemore et al., 1997; Glaser et al., 1998). Indirect personto-person or zoonotic transmission may occur by contamination of water used
for recreation (swimming pools) or drinking or by food (raw milk and meat, farmmade apple cider)(Casemore et al., 1997).
Waterborne outbreaks of cryptosporidiosis have been attributed to contaminated
drinking water, both from surface and ground water sources (Craun, 1990;
Mackenzie et al., 1994; de Jong & Andersson, 1997), and to recreational water
and swimming pools (Joce et al., 1991; MacKenzie et al., 1995; van Asperen et
al., 1996; Anon, 1998; Kramer et al., 1998).
Drinking-waterborne outbreaks have been caused by contamination of the
source water due to heavy rainfall or melting snow (Richardson et al., 1991;
Pett et al., 1993; MacKenzie et al., 1994) or to sewage contamination of wells
(d’Antonio et al., 1985; Kramer et al., 1996), inadequate treatment practices
(Richardson et al., 1991; Craun et al., 1998) or treatment deficiencies (Anon.,
1990; Leland et al., 1993; Craun et al., 1998) or combinations of these factors
(MacKenzie et al., 1994). Also, leakage or cross-connections in the distribution
system have caused outbreaks of cryptosporidiosis (Craun, 1990; de Jong &
Andersson, 1997; Craun et al., 1998). The number of people affected by a
16
Protozoan parasites
cryptosporidiosis outbreak through drinking water ranges from several up to
400.000.
During several of these outbreaks, oocysts were detected in the drinking water
in a wide range of concentrations (Haas & Rose, 1995). Examination of drinking
water during outbreaks is usually too late to determine the concentrations that
triggered the outbreak. To obtain ‘historical’ data on the occurrence of oocysts
in drinking water, researchers have attempted to detect oocysts in ice
(MacKenzie et al., 1994), in in-line filters (van Asperen et al., 1996) and in
sediments of water storage tanks (Pozio et. al., 1997). The detected
concentrations are probably an underestimation of the concentrations that led to
the outbreak, although Haas & Rose (1994) showed for the Milwaukee outbreak
that, with some assumptions, the measured concentration in drinking water was
close to the predicted concentration on the basis of the attack rate, water
consumption and dose-response relation.
Low oocyst concentrations in drinking water have also been found in situations
where no evidence for the occurrence an outbreak was present (LeChevallier et
al., 1991; Karanis & Seitz, 1996; Rose et al., 1997; McClellan, 1998). Current
detection methods do not allow the determination of pathogenicity of oocysts in
water, which makes it difficult to determine the significance of low oocyst levels
in drinking water. Given this uncertainty, detection of oocysts in treated water
should always lead to the use of additional tests to confirm the presence of
(viable) C. parvum oocysts and a thorough examination of other water quality
parameters that may indicate a faecal contamination event. If these additional
tests indicate the presence of C. parvum oocysts, this should lead to an
epidemiological study to determine if significant waterborne transmission occurs
and careful examination of the source of the contamination and the installation
of control measures (improved source protection and/or water treatment).
Giardia intestinalis
Taxonomy
Giardia is a flagellated protozoan. The taxonomy and host specificity of this
organism have been and still are matter of much debate. Giardia has been found
in more than 40 animal species (Meyer, 1994). Nowadays, five species of
Giardia are established in the scientific literature: including the three species
proposed by Filice (1952): G. muris in rodents, birds and reptiles, G. intestinalis
(syn: duodenalis, syn: lamblia) in mammals (including man), rodents, reptiles and
possibly in birds, G. agilis in amphibians, G. ardae in the Great Blue Heron
(Erlandsen et al., 1990) and G. psittaci in the budgerigar (Erlandsen & Bemrick,
1987). Recently, a morphologically distinct Giardia was isolated from the strawnecked ibis (Forshaw et al., 1992), that was later suggested to be a distinct
strain of G. ardae (McRoberts et al., 1996).
Giardia is thought to be predominantly asexual, which makes the species
concept difficult to apply.
17
Chapter 1
A high degree of genetic heterogeneity is found in human and animal isolates
(Nash et al., 1985; Andrews et al., 1989; Meloni et al., 1989; Morgan et al.,
1994) which makes speciation uncertain and suggests that it is a clonal parasite
(Tibayrenic, 1994). G. intestinalis can be subdivided by several techniques into
two groups (Homan et al., 1992, 1994). It is still uncertain if and how this
heterogeneity is related to host specificity and pathogenicity of Giardia.
Life-cycle
Giardia has a simple life cycle (Feely et al., 1990; Meyer, 1994). As with
Cryptosporidium, the parasite is shed with the faeces as environmentally robust
cyst, that is transmitted to a new host. In the duodenum of the new host, the
trophozoite emerges from the cysts and completes a mitotic division to produce
two trophozoites that attach to the epithelial cells by their adhesive disc and
feed on the epithelial cell. The trophozoites detach from the epithelial cells,
probably because these cells have a rapid turnover (72 hours) and undergo
mitotic division in the intestinal lumen. During periods of diarrhoea, these
trophozoites may be transported with the intestinal contents and are excreted.
The trophozoites do not survive long outside the host. During the passage
through the intestine, part of the trophozoites begin to encyst and leave the
host with the faeces as cysts. Cysts are more often encountered in formed
stools. Giardia intestinalis cysts are elliptical and 8-12 µm long and 7-10 µm
wide. The cyst wall is 0.3-0.5 µm thick and has a fibrillous structure. In the
cyst, two to four nuclei can be found together with axonemes of the flagella of
the trophozoite.
Pathogenicity
Human feeding studies with G. intestinalis cysts produced a dose response
relation between the probability of infection and the ingested cyst dose
(Rendtorff, 1954). No data on the viability of the ingested cysts were provided.
A dose of 10 cysts resulted in an infection in 100% (2/2) of the volunteers. The
dose-infection relation could be described with an exponential model (Rose et
al., 1991b): Pi = 1-e-0.0199*dose (95% CI of r: 0.0044-0.0566). Although overall
53% of the volunteers became infected in this feeding study, and changes in
bowel motions were observed, none of the volunteers developed symptoms of
giardiasis. The infection-to-illness ratio varies between different isolates, as
shown by the different response to two different isolates from symptomatic
human infections in the volunteer study of Nash et al. (1987). Also host factors
(age, nutritional status, predisposing illness, and previous exposure) determine
the outcome of an infection (Flannagan, 1992). Asymptomatic carriage appears
to be the most common form of infection with Giardia (Farthing, 1994), ranging
from 16-86% of the infected individuals. The mechanism by which Giardia
causes diarrhoea and malabsorption is still unclear. Giardia could act as physical
barrier, but the area covered by trophozoites is probably too small for affecting
the absorption of nutrients. No evidence has been found for the production of
toxins (Buret, 1994). Giardia infections appears to affect gut enzyme (lactase,
disaccharidase) activities and damage the mucosal surface (shortening of crypts
18
Protozoan parasites
and villi) and give rise to overgrowth of the small intestine by bacteria (Tomkins
et al., 1978) or yeasts (Naik et al., 1978).
The disease
The time between infection and the occurrence of Giardia cysts in the stool is
12 to19 days (Jokipii et al, 1985). The incubation period for the occurrence of
symptoms varies between 1-75 days, but is generally between 6-15 days, and
coincides with the occurrence of Giardia in stool (Rendtorff, 1954; Brodsky et
al., 1974). The most prominent symptoms are diarrhoea (fatty, yellowish)
weakness, weight loss and abdominal pain and to a lesser extent nausea,
vomiting, flatulence and fever. In the majority of cases, the infection is acute
and self-limiting within 2-4 weeks. A significant proportion of the infected
population will go on to have chronic infection with intermittent diarrhoea
(estimated at 30-50%)(Farthing, 1994). Weight loss can be profound (10-20%)
in this group. The ability of Giardia to change the surface epitopes of the
trophozoites during infection (Nash, 1992), may play a role in the occurrence of
chronic infections. There is evidence that infection with Giardia results in ‘failure
to thrive’ in children, by impairment of the uptake of nutrients (especially fats
and vitamin A and B12) (Farthing, 1994; Hall, 1994). Excretion of cysts varies
between 106-108 per gram of stool, as determined in positive stool samples
(Tsuchiya, 1931), but a significant proportion of the stool samples does not
show Giardia in detectable levels. Excretion patterns vary between hosts and
isolates.
Prevalence
Giardia infections are very common in children in developing countries (Rabbani
& Islam, 1994; Farthing, 1994). Also in developed countries, the prevalence
peaks at the age of 1-4 years (Flannagan, 1992); a second peak is observed at
the 20-40 age group, partly due to the care for the young children and partly
due to travelling. In developing countries, the prevalence of giardiasis in patients
with diarrhoea lies around 20%, ranging from 5-43% (Islam 1990). In developed
countries, this prevalence varies from 3% (Hoogenboom-Verdegaal et al, 1989;
Adam, 1991; Farthing, 1994; Kortbeek et al., 1994) to 7% (Quinn, 1971).
As a reaction to infection with Giardia, both a humoral and cellular immune
response is generated by the host. Secretory IgA and IgM appear to play a role
in clearance of the intestinal infection, by reducing the mobility of trophozoites
and preventing their adhesion to the mucosa (Farthing and Goka, 1987). The
immune response can also be seen in the serum antibodies. The immune
response can give some degree of protection against reinfection, as indicated by
lower attack rates in chronically exposed populations (Istre et al., 1984; Rabbani
& Islam, 1994). This protection is limited however, recurrence of symptomatic
infections, even after several infections, is common (Gilman et al., 1988; Wolfe
1992; Hall, 1994), which may be related to the antigenic variation shown by
Giardia (Nash, 1992).
Giardiasis can be treated with nitroimidazoles, quinacrine and furazolidone
(Boreham, 1994). For patients with persistent giardiasis several approaches can
19
Chapter 1
be taken, such as increasing duration and dose of drug admission, administering
an alternate drug or a combination of drugs.
Routes of transmission
Person-to-person faecal-oral transfer of Giardia cysts is the major route of
transmission of giardiasis, as indicated by the high prevalence in situations with
poor hygienic conditions in developing countries, in day-care centres and
nurseries (Black et al., 1977; Pickering & Engelkirk, 1990; van de Bosch, 1991)
and secondary spread to the house-hold in day care centre outbreaks (Black et
al., 1977). Foodborne outbreaks have been the result of contamination of food
by infected workers or household members (Osterholm et al., 1981; Islam,
1990; Thompson et al., 1990).
The role of animals in the transmission of human giardiasis is still controversial.
Although Giardia commonly occurs in pet, farm and wild mammals, there is no
unequivocal evidence that these Giardia have caused infections in humans
(Erlandsen, 1994). Giardia intestinalis isolates from animals and man may be
morphologically indistinguishable (Flannagan, 1992) and this has led to many
reports on animal sources of human giardiasis, including waterborne cases
caused by Giardia cysts from beavers and muskrats (Moore et al., 1969; Dykes
et al., 1980). However, the genetic diversity within and between human and
animal isolates (Thompson et al., 1988) is too high to draw definite conclusions
regarding host specificity. Cross-transmission studies have not been well
controlled and the results have been contradicting (Davies & Hibler, 1979;
Hewlett et al., 1982; Belosevic et al., 1984; Kirkpatrick & Green, 1985; Woo &
Patterson, 1986).
Waterborne outbreaks of giardiasis have been reported for almost 30 years
(Moore et al., 1969; Brodsky et al., 1974; Craun, 1990). In the US, Giardia is
the most commonly identified pathogen with more than 100 waterborne
outbreaks, based on epidemiological evidence (Craun, 1990). Waterborne
outbreaks have also been reported from Canada, Australia, New Zealand, United
Kingdom and Sweden. These outbreaks have been linked to consumption of
untreated surface water that was contaminated by human sewage (Craun,
1990) or by wild rodents (Moore et al., 1969; Dykes et al., 1980), to ground
water that was contaminated by human sewage or contaminated surface water,
to surface water systems receiving only disinfection (Craun, 1984; Kent et al.,
1988) or ineffective filtration (Dykes et al., 1980; Craun, 1990) and by crossconnections or damage in the distribution system (Craun, 1986).
Cyclospora sp.
Taxonomy
Cyclospora was first described by Eimer in 1870 from the intestines of moles, and
is related taxonomically to other protozoan parasites such as Cryptosporidium and
Toxoplasma. The first likely observation of this parasite as a pathogen for human
beings was by Ashford (1979). Confirmation of the coccidian identity and genus
was made in 1993 (Ashford et al., 1993; Ortega et al., 1993). Cyclospora is a
20
Protozoan parasites
member of the subphylum Apicomplexa, class Sporozoasida, subclass Coccidiasina,
family Eimeriidae. Organisms of the genus Cyclospora have an oocyst with two
sporocysts, each of which contains two sporozoites (Levine, 1973) and have
been found in snakes, insectivores and rodents. Molecular phylogenetic analysis
suggests that the genus Cyclospora is closely related to the genus Eimeria (Relman
et al., 1996).
Life-cycle
Many of the details of the life cycle of Cyclospora in human beings are not yet
known. Cyclospora completes its life cycle within one host (monoxenous). Ortega
et al (1993) proposed that Cyclospora that are infective to human beings should be
designated Cyclospora cayetanensis on the basis of the development of the oocyst
in vitro. However, Ashford et al., (1993) question the species name and Bendall et
al (1993) preferred the use of the term CLB (denoting Cyclospora-like body) until
further information is forthcoming regarding the biology of this coccidian parasite. In
this review, Cyclospora sp. will be the nomenclature used to describe those
organisms infective to man.
The endogenous stages of Cyclospora sp. are intra-cytoplasmic and contained
within a vacuole (Bendall et al., 1993), and the transmissive stage, the oocyst, is
excreted in the stool. The life cycle of Cyclospora sp. may complete within
enterocytes (Sun et al., 1996). Cyclospora sp. oocysts are spherical, measuring 810 µm in diameter. They are excreted unsporulated in the stool and sporulate to
infectivity in the environment. Unsporulated oocysts contain a central morula-like
structure consisting of a variable number of inclusions whilst sporulated oocysts
contain two ovoid sporocysts. Within each sporocyst reside two sporozoites.
Each sporozoite measures 1.2 x 9 µm.
Pathogenicity
Cyclospora sp. infects enterocytes of the small bowel and can produce disease
(Bendall et al., 1993). Both symptomatic and asymptomatic states have been
described. A moderate to marked erythema of the distal duodenum can occur
with varying degrees of villous atrophy and crypt hyperplasia (Connor et al.,
1993). However, little is known of the pathogenic mechanisms. As yet, no
virulence factors have been described for Cyclospora sp. No animal or human
feeding studies have been undertaken. As for Giardia and Cryptosporidium, it is
assumed that the organisms are highly infectious, and that doses less than 100
sporulated oocysts may lead to a high probability of infection.
The disease
Symptoms include watery diarrhoea, fatigue, abdominal cramping, anorexia,
weight loss, vomiting, low-grade fever and nausea which can last several weeks
with bouts of remittance and relapse. The incubation period is between 2 and
11 days (Soave, 1996) with moderate numbers of unsporulated oocysts being
excreted for up to 60 days or more. Illness may last for weeks and episodes of
watery diarrhoea may alternate with constipation (Soave, 1996). In
immunocompetent individuals the symptoms are self-limiting and oocyst
21
Chapter 1
excretion is associated with clinical illness (Shlim et al., 1991), whereas in
immunocompromised individuals diarrhoea may be prolonged.
Prevalence
Cyclospora sp. oocysts have been isolated from the stools of children,
immunocompetent and immunocompromised adults. Oocysts have been described
in the stools of residents in, and travellers returning from, developing nations, and
in association with diarrhoeal illness in individuals from North, Central and South
America, the Caribbean, the Indian sub-continent, Southeast Asia, Australia and
Europe. Outbreaks of cyclosporiasis have been reported from Nepal and North and
South America. In north America and Europe cyclosporiasis is associated with
overseas travel and travellers’ diarrhoea. Point source outbreaks have been reported
in the USA and Nepal. In 1996, a total of 1465 cases were reported in the USA and
Canada, with around half of them occurring following events at which raspberries
had been served (Anon., 1996; Herwaldt et al., 1997). Most cases occurred
during spring and summer. Sporadic cases of cyclosporiasis have been reported
from many countries and Cyclospora sp. oocysts are increasingly being identified in
stools from immunocompetent individuals without foreign travel histories. Studies in
Nepal, Peru and Tanzania seek to address Cyclospora sp. epidemiology, life cycle
and pathology.
Cyclospora sp. oocysts were detected in faecal samples from 11% of Haitians
with chronic diarrhoea who were seropositive for human immunodeficiency virus
(HIV) (Pape et al., 1994). Apart from HIV, Cyclospora sp. oocysts were the sole
pathogen identified in many of these patients. Whilst clinical disease can resolve
without treatment, trimethoprim-sulphamethoxazole (TMP-SMZ) is the drug of
choice.
Routes of transmission
Epidemiology indicates that Cyclospora sp. is transmitted by water and food
(Hoge et al., 1993; Anon., 1996; Herwaldt et al., 1997). An outbreak amongst
house staff and employees in a hospital dormitory in Chicago occurred following
the failure of the dormitory’s water pump. Illness was associated with the
ingestion of water in the 24 hours after the pump failure and Cyclospora sp.
oocysts were detected in the stools of 11 of 21 persons who developed
diarrhoea (Anon, 1991; Wurtz, 1994).
An outbreak occurred amongst British soldiers and dependants stationed in a
small military detachment in Nepal and 12 of 14 persons developed diarrhoea.
Cyclospora sp. oocysts were detected in stool samples from 6 of 8 patients.
Oocysts were also detected microscopically in a concentrate from a 2 litre water
sample. Drinking water for the camp consisted of a mixture of river water and
chlorinated municipal water. Chlorine residuals of 0.3 to 0.8 ppm were
measured before and during the outbreak. No coliforms were detected in the
drinking water (Rabold et al., 1994).
DETECTION METHODS
22
Protozoan parasites
Cryptosporidium and Giardia
The methodology required for the detection of Cryptosporidium oocysts and
Giardia cysts in water is completely different from that traditionally used in the
water industry. The methods that are currently available are at best tentative
because of their low and variable recovery and the inability to differentiate
viable oocysts of strains that are infectious to humans. The overall procedure
consists of several stages, namely: sample collection and concentration,
separation of (oo)cysts from contaminating debris and detection of (oo)cysts.
Many factors, such as water quality and age of the (oo)cysts, can have
significant effect on the overall recovery efficiency and thus it is almost
impossible to compare the effectiveness of two methods that have been
performed in different laboratories unless these factors are standardised.
Furthermore, there is considerable interest in determining if (oo)cysts are viable
and potentially infectious. Thus methods have been and are currently being
developed to assess the viability of (oo)cysts in the environment.
Quality assurance
Microscope counts
Care must be taken to ensure that the particles being counted are (oo)cysts,
whether or not they contain sporozoites, and that algae and yeast cells are
excluded from any counts that are made. The criteria used for determining that
a particle is in fact a Cryptosporidium oocyst or Giardia cyst vary between
laboratories. Some workers use only the fact that (oo)cysts fluoresce when
labelled with a fluorescein isothiocyanate tagged anti-Cryptosporidium or antiGiardia monoclonal antibody and that it is in the proper size range that a particle
is a cyst or oocyst, whilst others will additionally use differential interference
contrast microscopy or nucleic acid stains to determine if the particles that are
counted are indeed (oo)cysts. This more detailed analysis allows the
confirmation of the counted particles as presumptive (oo)cysts.
Many factors influence the microscope counts: the amount of background
debris and background fluorescence, experience and alertness of the counting
technician, fluorescence intensity after staining with the monoclonal antibody
and the quality of the microscope. QA protocols should define how these
factors are addressed.
Recovery efficiency
Given the low and variable recovery efficiency of the methods that are used for
environmental monitoring for Cryptosporidium and Giardia, it is essential that
laboratories collect their own data on the recovery efficiency of their method in
the different water types they monitor. This can be achieved by seeding a
second water sample with a known number of cysts and oocysts and determine
which percentage of these (oo)cysts is recovered by the total protocol for
sampling, processing and counting of environmental samples.
This assay is influenced by the number, age and storage conditions of the
(oo)cysts used for seeding. These have to be standardised (at least within a
laboratory) to collect meaningful recovery data. The recovery efficiency should
be assessed sufficiently frequent to be able to determine how the variation in
23
Chapter 1
the recovery efficiency influences the uncertainty of the monitoring data. This is
essential for the interpretation of environmental monitoring.
Concentration techniques
Cartridge filtration
The initial methodology to detect Giardia and Cryptosporidium in water used
polypropylene cartridge filters with a nominal pore size of 1 µm, through which
large volumes of water (100-1000 litres) are passed at a flow rate of 1-5 litres
per minute. Trapped material is then eluted by cutting the filter open and
washing either by hand or by stomaching using a dilute detergent solution. The
resulting washings from these cartridges sometimes totals three or four litres
and they must then be further concentrated by centrifugation. The ability to
recover Cryptosporidium oocysts by this technique was originally reported to be
in the range of 14-44% (Musial et al., 1987) although lower recovery
efficiencies (<1-30%) have often been reported since (Ongerth & Stibbs, 1987;
Clancy et al., 1994; Shepherd & Wyn-Jones, 1996). Differences in reported
recovery rates may be due to a number of factors including water quality,
laboratory efficiency and oocyst age.
Membrane filtration
A method described by Ongerth & Stibbs (1987) utilised large (142 or 293 mm
diameter) 2 µm absolute, flat bed membranes for the concentration of oocysts
from water samples and many workers have now adopted this procedure. Water
is pumped through the membranes and the concentrated materials are recovered
by ‘scraping’ the surface of the membrane together with washing with dilute
detergent followed by further concentration using centrifugation. However,
whilst with low turbidity water, it is relatively easy to filter 10-40 litres, with
some high turbidity waters, it is only possible to filter 1-2 litres. As with
cartridge filtration, a range of recovery efficiencies has been reported for flat
bed membranes. Nieminski et al. (1995) reported an average recovery of 9% for
Cryptosporidium and 49% for Giardia. In a study of the efficiencies of several
different membranes for recovering both Cryptosporidium oocysts and Giardia
cysts, Shepherd & Wyn-Jones (1996) suggested that 1.2 µm cellulose acetate
membranes gave higher recovery (30-40% and 50-67% respectively) than the 2
µm polycarbonate membranes (22-36% and 41-49% respectively) preferred by
Ongerth & Stibbs (1987).
Flocculation
Another established procedure for concentrating (oo)cysts is the calcium
carbonate flocculation procedure developed by Vesey et al. (1993b). A fine
precipitate of calcium carbonate (CaCO3) is formed in a water sample by the
addition of calcium chloride and sodium bicarbonate, followed by adjusting the
pH to 10.0 with sodium hydroxide. After allowing the precipitate to settle, the
supernatant fluid is aspirated off and the sedimented material resuspended after
dissolving the calcium carbonate with sulphamic acid. Recovery efficiencies
using this method have been reported to be as high as 70% for both
Cryptosporidium and Giardia (Campbell et al., 1994; Vesey et al., 1993b; Vesey
24
Protozoan parasites
et al., 1994; Shepherd & Wyn-Jones, 1996). More recent work has
demonstrated that this is the upper limit of the detection efficiency and that
lower recoveries are usually encountered. Use of aged oocysts for seeding
experiments together with leaving the oocysts in contact with water for a few
days prior to analysis showed that recovery rates of 30-40% were more
normally seen. The viability of the oocysts is affected by this concentration
(Campbell et al., 1995). Flocculation with aluminium sulphate (Al2(SO4)3) did not
affect the viability of oocysts, while the recovery efficiency was comparable to
the CaCO3 flocculation (Schwartzbrod, pers. comm.).
New methods
The search for new methods for concentrating water samples to detect the
presence of protozoan parasites continues and many methods have been
evaluated, including cross-flow filtration, continuous flow centrifugation and
vortex flow filtration (Whitmore, 1994). Methods which are currently receiving
attention include vortex flow filtration (Fricker et al., 1997), the Gelman
envirochek filters (Clancy et al., 1997) and the Genera filter system (Sartory,
pers. comm.), amongst others.
There continues to be much debate over which method is most appropriate.
Realistically there is no one single method which is most suitable for all
situations. The choice of method should be made with due regard to a number
of factors, including the purpose of sampling, the water quality and the facilities
in the laboratory which will perform the analysis. Ideally, the method chosen
should efficiently concentrate as large a sample as possible and yield a
concentrate which can be examined easily. Many workers prefer to concentrate
only a small volume of water initially and to examine the entire concentrate,
whilst others take large samples and examine only a fraction of the final
concentrate. Either approach is defensible, but the methods used to concentrate
small volumes (e.g. 10-20 l) tend to be easier to perform and generally have a
higher recovery efficiency and so it is often preferable to take a large number of
low volume samples and to examine all of the concentrate. Other factors which
may affect the choice of concentration method include the site of sample
collection and the distance which samples must be transported.
Separation techniques
Since the concentration of Cryptosporidium oocysts and Giardia cysts is based
almost exclusively on particle size, the techniques are not specific and a large
amount of extraneous material is concentrated as well. This material may
interfere with the successful detection of (oo)cysts, either by increasing the
total volume which needs to be examined, or by obscuring or mimicking
(oo)cysts during examination. Some form of separation technology is therefore
normally required to reduce the time taken to examine a sample and to prevent
(oo)cysts being missed.
25
Chapter 1
Density centrifugation
Density centrifugation is used by many workers to separate (oo)cysts from
background debris and thus reduce the amount of material to be examined.
Several workers use sucrose density centrifugation to separate parasites from
faecal material in clinical samples. This basic technique has been adopted for
use with environmental samples, although some workers prefer to use Percollsucrose or Percoll-percoll gradients. Whatever flotation method is used, several
groups have demonstrated that this is an inefficient procedure when trying to
detect protozoan parasites in water concentrates. Of particular interest was the
finding of Bukhari & Smith (1996) that sucrose density centrifugation selectively
concentrated viable, intact Cryptosporidium oocysts. Fricker (1995)
demonstrated that the recovery of oocysts from water samples could be
affected by the length of time that they were in contact with the water
concentrate but that this was only the case when sucrose flotation was
performed. Spiked samples which are examined directly without density
centrifugation gave similar recovery efficiencies, irrespective of whether they
were examined immediately after seeding or after 48 hrs contact with the
concentrate. However, when sucrose flotation was used, the recovery of
(oo)cysts in raw water fell from a mean of 55% to 18% after the same period of
contact. This reduction in recovery efficiency was also seen with concentrates
of reservoir water (67 to 23%) and fully treated water (80 to 52%).
Immunomagnetic separation
Autofluorescing algae, which may not be completely removed by the density
gradient centrifugation, can cause severe problems when examining slides for
protozoa by epifluorescence microscopy. More efficient methods for separation
of (oo)cysts from other particulates have been sought. Many workers have
attempted the use of immunomagnetic separation (IMS). The principles behind
this technology involve the attachment of specific antibodies to magnetisable
particles and efficient mixing of the particles in the sample. The (oo)cysts attach
to the magnetisable particles and are isolated from this debris with a strong
magnet. The technique is very simple, but there are several sources of failure.
An important source is the quality and specificity data of the monoclonal
antibodies which are available. Most of the commercially available monoclonal
antibodies to Cryptosporidium or Giardia are of the IgM type, and are therefore
of low affinity since they have not undergone affinity maturation or isotype
switching. When IMS is used and beads are mixed with water concentrates, the
immunoglobulin-(oo)cyst-bonds are subjected to shear forces and therefore the
stronger the bond, the more likely the bead is to remain in contact with the
(oo)cyst. The way in which the antibody is attached to the bead may also have
an effect on recovery efficiency, since if the attachment between the bead and
the antibody is not strong, the antibody may detach and the oocyst will not be
recovered. The turbidity of the water concentrate appears to be the most critical
factor associated with the recovery efficiency of IMS. Oocysts seeded into
relatively clean suspensions are recovered efficiently, with recoveries of over
90% being reported (Campbell et al., 1997a,b). However, the real benefit of a
good separation technique is with samples which have yielded a highly turbid
26
Protozoan parasites
concentrate and it is in these samples that IMS does not appear to perform as
efficiently. The use of antibodies of higher affinity may serve to improve the
recovery efficiency of oocysts from high turbidity samples. Although this
technique is also able to separate Giardia cysts, not much effort has been put
into testing the recovery efficiency of these cysts by IMS.
Flow cytometry
Workers in the United Kingdom attempted to use flow cytometry with
environmental samples in order to detect Cryptosporidium oocysts, but found
that the sensitivity of these instruments was not high enough to distinguish
oocysts from background noise (Vesey et al., 1991). Incorporation of a cell
sorting facility onto flow cytometers enabled oocysts to be sorted efficiently
from background material (Vesey et al., 1993a). This technique is shown to
work equally efficient for Giardia cysts (Vesey et al., 1994; Medema et al.,
1998a). Water concentrates are stained in suspension with FITC-labelled
antibodies and passed through the fluorescence activated cell sorter (FACS).
Particles with the fluorescence and light scatter characteristics of (oo)cysts are
sorted from the sample stream and collected on a microscope slide or membrane
filter, that is examined by epifluorescence microscopy to confirm the presence
of (oo)cysts. The FACS procedure is not specific and sensitive enough to enable
the count of sorted particles as a definitive number of (oo)cysts present, since
other organisms/particles of similar size may cross-react with the monoclonal
antibody and have similar fluorescence characteristics. In addition, some water
samples contain high numbers of autofluorescent algae which may also mimic
(oo)cysts and therefore lead to incorrect conclusions if the FACS is used directly
to produce (oo)cyst counts. However, the confirmation by epifluorescence
microscopy can be performed much easier and more reliably than direct
microscopy of non-sorted samples. Several researchers from the United States,
France and the Netherlands have confirmed the benefits of FACS when
examining water samples for the presence of (oo)cysts (Danielson et al., 1995;
Compagnon et al., 1997; Medema et al., 1998a). FACS is widely used in the
United Kingdom for water analysis and is becoming more and more adopted in
other parts of Europe, in Australia and in South-Africa.
Detection
Immunofluorsecence microscopy
Routine detection of Cryptosporidium oocysts and Giardia cysts relies on the
use of epifluorescence microscopy which may be applied to examine material
deposited on multiwell slides or membrane filters. The (oo)cysts are specifically
stained with monoclonal antibodies which have been labelled directly with FITC
or are labelled during staining with an FITC-labelled anti-mouse antibody. There
have been no definitive studies to compare the efficiency of these procedures,
but the tendency now is towards staining with a directly labelled antibody. This
tends to give less non-specific binding and can make preparations easier to
examine. Several anti-Cryptosporidium antibodies and anti-Giardia antibodies are
commercially available and whilst most workers have their preferences, there
does not appear to be a single antibody which is preferred for all purposes. One
27
Chapter 1
specific failing of the commercially available antibodies is that they all apparently
cross-react with other members of the genera and therefore cannot be used to
specifically identify C. parvum or G. intestinalis.
A number of other detection techniques have been tried by various workers in
order to improve the ease of identification of both Cryptosporidium oocysts and
Giardia cysts.
FISH
Fluorescence In-Situ Hybridisation (FISH) has been suggested as a tool for the
specific detection of Cryptosporidium parvum (Vesey et al., 1997; Lindquist,
1997). Vesey et al. (1997) also showed that the stainability of oocysts with the
FISH-method correlated with excystation. This FISH method could be combined
with the IFA method. However, the intensity of the FISH-fluorescence signal is
relatively weak, which makes microscopic interpretation difficult.
PCR
Perhaps one of the most extensively tested procedures is the use of the
polymerase chain reaction (PCR) to detect specific sequences of nucleic acids
which may be species or genus specific. Clearly, the ability to distinguish
between C. parvum and other morphologically similar members of the genus is
useful and nucleic acid based techniques may prove useful for this.
However, despite the exquisite specificity and sensitivity which PCR can offer,
difficulties have been experienced with the application of PCR to water
concentrates. This has largely been due to inhibition of the DNA amplification
process. PCR is sensitive to the concentration of many compounds within the
reaction mixture and those of particular concern to researchers working with
water concentrates are divalent cations and humic and fulvic acids, which are
compounds frequently found in water and which can cause a high degree of
inhibition. Nonetheless many workers have described protocols for the detection
of Cryptosporidium oocysts by PCR and a wide variety of primers have been
described. These primers have been designed from various regions of the
genome and some which have apparent specificity include those from regions
coding for the 18 S rRNA (Johnson et al., 1995), or mRNA coding for the
Cryptosporidium heat shock protein Hsp70 (Stinear et al., 1996, Kaucner &
Stinear, 1998), in combination with cell culture (Rochelle et al., 1996, 1997).
Abbaszadegan et al. (1997) first reported the application of PCR primers from
gene sequences coding for inducible heat shock proteins to specifically detect
Giardia cysts. The sensitivity of the standard PCR was reported to be one cyst
in water samples. They also reported that amplification of heat shock-induced
mRNA utilising the same HSP primers was indicative of viable Giardia cysts.
The use of PCR for the detection of cysts and oocysts in water concentrates
offers some advantages over that of direct microscopical examination, since the
process can largely be automated and thus several samples can be processed
simultaneously. Furthermore, the technique is theoretically sensitive down to a
level of a single (oo)cyst and recent developments have suggested that it may
be possible to distinguish viable from non-viable (oo)cysts. Some workers claim
28
Protozoan parasites
to be able to detect a single oocyst in a water concentrate by using a procedure
involving reverse transcription (RT) PCR where the target sequence codes for
the Cryptosporidium heat shock protein Hsp 70 (Stinear et al., 1997). The data
presented showed that a single viable oocyst could be detected by this
procedure, even in the presence of PCR inhibitors. Such a method would be of
considerable value to the water industry, facilitating rapid screening of samples
although as yet it is not quantitative and thus may be of limited value in some
circumstances.
The use of RT-PCR against induced mRNA, a nucleic acid with a short half-life,
overcomes the concern that “false positive” results could be obtained either
from non-viable oocysts or from free DNA. Many researchers still favour a
holistic approach, where the intact organism can be viewed directly. A
combined approach may be used whereby molecular techniques are used as a
screening tool on a portion of a water concentrate and that where positive
results are generated, other approaches which involve microscopical
examination are used.
Methods for determining oocyst viability
The significance of finding oocysts in treated and to a lesser extent raw waters
is not always clear, since some of the organisms which are detected may be
non-viable and thus pose no threat to public health. Therefore, there has been
considerable interest in developing in vitro methods which can determine oocyst
viability.
Excystation
The most widely accepted in vitro procedure for determining oocyst viability,
excystation, has not been used in combination with the IFA method, because
excystation is difficult to incorporate in the IFA protocol. Excystation has been
used in combination with PCR to detect the presence of viable Cryptosporidium
oocysts (Filkorn et al., 1994; Wiedenmann et al., 1997). The sensitivity of this
method in environmental samples needs further research. Excystation has been
used in survival and disinfection studies. In the latter, this technique appears to
yield a lower inactivation rate than the neonatal mouse infectivity assay (Finch
et al., 1993a; Clancy et al., 1998).
Vital dyes
The ability of Giardia cysts to stain with the vital exclusion dye propidium iodide
(PI) has been shown by various workers to correlate with the inability to excyst
or infect animals (Schupp & Erlandsen, 1987; Smith & Smith, 1989). PI can
therefore be used as indicator of cell death for Giardia cysts.
Campbell et al. (1992) developed a procedure based on the exclusion of PI for
Cryptosporidium oocysts, using 4'6-diamidino-2-phenyl indole (DAPI) as
supporting stain, which gave a good correlation with in vitro excystation. Four
classes of oocysts can be identified using the assay: those which are viable and
include DAPI but exclude PI, those which are non-viable and include both DAPI
and PI and two classes which include neither DAPI or PI, those with internal
contents (sporozoites) and therefore potentially viable, and those without and
29
Chapter 1
therefore non-viable, as determined by DIC. microscopy. The DAPI/PI procedure
is simple to perform and whilst some workers have expressed some reservations
over its' applicability, it can be used for routine environmental work. The
incorporation of DAPI into the nucleic acid acts as a further criterion for
determining if a particle is an oocyst or not.
An alternative to the DAPI/PI approach to determine viability has been
suggested by Belosevic and Finch (1997) who have used new nucleic acid
stains to differentiate between viable and non-viable oocysts. Two new stains
have been identified, SYTO9 which stains non-viable oocysts green or bright
yellow, while viable oocysts have a green halo surrounding the cell whilst the
interior remains unstained and MPR71059 which stains non-viable oocysts red
whilst viable oocysts remain unstained. These approaches have not been widely
tested although Belosevic and Finch (1997) have demonstrated that the results
obtained with these dyes, correlate well with mouse infectivity using an outbred
CD-1 neonatal mouse model. Since these vital stain-assays are apparently
simple and quick to perform, they may be suitable for incorporation into the
methods for the detection of oocysts in water samples, but this has yet to be
proven.
Cell culture
Attempts have been made to develop in vitro models of infectivity using tissue
culture (Upton et al, 1994, Rochelle et al, 1996; Slifko et al., 1997). For these
assays, water samples are concentrated by normal procedures and bacteria may
be removed by exposure of the concentrate to concentrations of chlorine which
are lethal to bacterial cells but which are thought not to effect oocysts. The
concentrates are then inoculated onto the tissue culture monolayer, left in
contact for a period to allow potentially infectious oocysts to infect cells before
the remaining debris is washed away. The monolayer is then left for 24-48
hours before being examined for the presence of intracellular parasite antigen or
nucleic acid. Immunofluorescent techniques have been used to identify cells
which have become infected. This offers a way in which infection may
potentially be quantified. However, it is not clear if the presence of a single
infectious oocyst will lead to one or more infected cells. In theory one might
expect that an oocyst which excysts successfully would produce four infected
tissue culture cells, but initial results have not demonstrated that this can be
consistently achieved. Other workers (Rochelle et al., 1996) have adopted a
somewhat different approach whereby they detect the presence of
Cryptosporidium nucleic acids using PCR. Whilst the cell culture method cannot
be used to directly enumerate the oocysts present in any given sample, it can
be applied in a "most probable number" format to give an estimation of the
number of oocysts present in a water concentrate.
Molecular methods
The RT-PCR methods that amplify induced mRNA that codes for heat shock
proteins also indicate viability of Giardia cysts (Abbaszadegan et al., 1997) and
Cryptosporidium oocysts (Stinear et al., 1997; Kaucner & Stinear, 1998). In
30
Protozoan parasites
combination with the reported sensitivity and specificity (see Detection), these
methods may prove to be very valuable for the water industry.
Typing methods
With the current detection techniques, it is not possible to identify the origin of
(oo)cysts in a water sample. Several typing methods are available for both
Cryptosporidium and Giardia and these are able to discriminate between human
and animal C. parvum strains (Ogunkadale et al., 1993; Bonnin et al., 1996;
Deng & Cliver, 1998), but these are not yet applicable to surface water
samples.
Cyclospora
Detection methods for stool samples
No methods have been developed for the detection of Cyclospora in
environmental samples. Therefore, the information on detection of this parasite
in stool samples is given as guidance.
Identification of Cyclospora in stool samples is based upon the appearance of
the oocyst either in direct or concentrated wet films. Concentration either by the
formalin-ether (formalin-ethyl acetate) method or sucrose flotation is effective.
Oocysts have also been reported from jejunal aspirates (Bendall et al., 1993).
Organisms seen in stool samples are normally the unsporulated oocysts of
Cyclospora sp. In wet mounts, oocyst walls appear as well-defined nonrefractile spheres measuring 8-10 µm in diameter by bright field microscopy, and
within an oocyst is a central morula-like structure containing a variable number
of inclusions. At higher (x 400) magnification, the inclusions appear refractile,
exhibiting a greenish tinge. Oocysts are remarkably uniform in size (Ashford,
1979; Long et al., 1991). Occasionally, oocysts which either have collapsed
into crescents or are empty are encountered. Under UV illumination (330-380
nm) the oocyst wall autofluoresces causing the organisms to appear as blue
circles. Organisms do not stain with Lugol’s iodine. Staining of air-dried faecal
smears with acid fast stains can aid identification, and, according to Wurtz
(1994), the rapid dimethyl sulphoxide-modified acid fast staining method is more
effective than either the Kinyouin or the modified Ziehl-Neelsen method. Oocysts
stain variably with acid fast stains ranging from deep red to unstained. A
modified safranin method (microwaving followed by safranin staining) stains
oocysts a brilliant reddish orange (Visvesvara et al., 1997).
Sporulated oocysts contain two sporocysts and each sporocyst contains two
crescentic sporozoites. In instances where excystation in vitro have been
successful, exposure of oocysts/sporocysts to an excystation medium at 37°C
for up to 40 minutes causes the emergence of two crescentic sporozoites from
each sporocyst.
Concentration techniques for environmental samples
As mentioned earlier, no method has been developed specifically for the
detection of Cyclospora sp. in environmental samples, but because Cyclospora
31
Chapter 1
sp. oocysts are larger than C. parvum oocysts and smaller that G. intestinalis
cysts, it is assumed that methods developed for Cryptosporidium and Giardia
will prove effective for sampling and recovering Cyclospora sp. oocysts from
water concentrates.
Detection techniques for use in environmental samples
There are no in vitro culture methods for increasing the numbers of Cyclospora
sp. oocysts nor have any in vivo amplification models been described. A
proportion of oocysts stored in faeces, water or 2.5% potassium dichromate at
temperatures between 22°C and 37°C for up to 14 days in the laboratory will
sporulate (Ortega et al., 1993; Smith et al., 1997). No commercially available
polyclonal or monoclonal antibody with specificity to exposed epitopes on
Cyclospora sp. oocysts is available currently. Therefore, the autofluorescent
properties of the oocyst wall under UV illumination have been used in an attempt to
detect oocysts in a variety of food and water concentrates. The primers identified
by Relman et al. (1996), which amplify the small subunit rRNA coding region, have
been used to amplify the Cyclospora-specific sequence from nucleic acid liberated
from berries (strawberries and raspberries) implicated in a series of outbreaks in the
USA in 1996. However, to date, no positive results have been reported.
CONTROL OF WATERBORNE TRANSMISSION
Cryptosporidium and Giardia are ubiquitous in surface waters throughout the
world. Reported concentrations generally range from 0.01-100 per litre. These
concentration data are not corrected for the (low) recovery of the detection
method, so the actual concentrations may be more than tenfold higher. Higher
concentrations are found in urbanised or agricultural waters than in pristine
waters (LeChevallier et al., 1991; Rose et al., 1991a).
Sources of surface water contamination are the discharge of untreated and
treated sewage, run-off of manure and wildlife. The relative significance of
these sources may differ between watersheds. Large rivers and lakes often
receive both agricultural run-off and treated and untreated domestic wastewater
and their relative contribution has not been quantified.
Wildlife may be an important contamination source in pristine watersheds and
has been implicated as the source of waterborne giardiasis, although this is still
a matter of much controversy.
Oocysts and cysts can survive for months in surface water (DeReignier et al.,
1989; Robertson et al., 1992; Chauret et al., 1995; Medema et al., 1997a).
Under natural conditions, the die-off rate of Cryptosporidium oocysts in water is
0.005-0.037 10log-units per day. For Giardia, the die-off rate is higher and
(more) temperature dependant: from 0.015 10log units per day at 1°C to 0.28
10
log-units per day at 23°C (DeReignier et al., 1989).
Although the state in which (oo)cysts occur in water (suspended or attached to
particles) is relevant for water treatment (sedimentation, filtration), and cysts
and oocysts readily attach to particles (Medema et al., 1998b), little information
32
Protozoan parasites
is available as yet on the significance of these factors in the environmental
ecology of (oo)cysts.
Recent information shows that overall 12% of groundwater supplies in the US
were contaminated with Cryptosporidium and/or Giardia (Hancock et al., 1997),
mostly in infiltration galleries and horizontal wells. No data on the level of
protection and travel time and distance of these groundwater sources were
given.
Prevention of the transmission of protozoan parasites through drinking water
requires a multiple barrier approach: protection of watersheds used for drinking
water production to contamination with protozoa and the installation of
adequate treatment coupled with verification that the treatment works
effectively by monitoring of water quality and operational parameters.
Watershed protection
The major sources of surface water contamination with Cryptosporidium and
Giardia are discharges of treated or untreated sewage (stormwater overflows),
run-off or discharges of manure from agricultural lands and, in more pristine
waters, wildlife. One of the most important aspects of watershed protection is
the recognition of the local sources of contamination with Cryptosporidium and
Giardia and to control the contamination as much as possible, by diversion or
treatment of discharges, reduction of direct input of faeces, especially in
otherwise pristine waters, by man, farm animals, wildlife or manure.
Treatment of sewage in activated sludge systems or waste stabilisation ponds is
an important barrier against environmental transmission. Both types of
processes remove 90-99.7% of the cysts and oocysts (Sykora et al., 1991;
Grimason et al., 1992).
Treatment of agricultural wastes before land application also reduces the
number and viability of Cryptosporidium oocysts: aerobic treatment of cattle
slurry at increased temperatures and ammonia concentrations rapidly inactivates
oocyst (Svoboda et al., 1997) and also composting of bedding reduces the
viability of oocysts.
Storm runoff and snowmelt from unprotected watersheds have been implicated
as source of peak contamination of source water (Stewart et al., 1997;
Atherholt et al., 1998), and may result in a treatment overload and the
contamination of drinking water with (oo)cysts. Knowledge of the
characteristics of the plume of contamination from watershed sources can be
used to locate and design abstraction points. The importance of this is
illustrated by the fact that the intake of the southern plant of Milwaukee in Lake
Michigan proved to be exactly in the plume of the Milwaukee river. The turbidity
in the raw water peaked and this coincided with treatment failure resulting in
the breakthrough of turbidity and oocysts in the Milwaukee drinking water
leading to the massive outbreak (MacKenzie et al., 1994).
Installation of pretreatment storage reservoirs flattens peak contaminations
(Ketelaars et al., 1995) and, because of the storage capacity, it is possible to
stop the intake of surface water temporarily during high contamination events.
33
Chapter 1
Since the protozoa are typically related to faecal contamination of surface
water, several studies have tried to determine the use of indicator bacteria to
predict high protozoa levels. No consistent relation is observed, however,
between indicator bacteria (thermotolerant coliform) levels and concentration of
Giardia or Cryptosporidium. The low and varying recovery of the protozoa
detection methods may be an important confounder in detecting these
relationships. As (oo)cysts are much more persistent than coliforms and
enterococci in water, it is likely that these bacteria are not valid indicators,
especially if the contamination source is distant. More persistent bacteria
(spores of Clostridium perfringens) may prove useful indicators for these
persistent protozoa (Payment & Franco,
1993; Hijnen et al., 1997). Since no valid surrogates are available, watershed
monitoring to determine local sources of contamination and to define the
amount of treatment necessary should therefore include monitoring for
protozoa.
Development of transport and fate models for predicting the (oo)cyst
concentrations based on data on the sources may help identify important
sources or environmental events that determine protozoa levels at abstraction
points (Medema et al., 1997b).
Currently, neither the number of species of Cyclospora infective to human beings is
known nor is it known whether human-derived oocysts are infectious to non-human
hosts. However, the primary sources of pollution will be human faeces
contaminated with oocysts. As Cyclospora sp. oocysts are larger than C. parvum
oocysts but smaller than G. intestinalis cysts, it is likely that they will be
discharged with final effluents from waste stabilisation ponds and sewage
treatment works. Oocysts take up to 14 days to mature (sporulate) in the
laboratory, sporulating more rapidly at higher (up to 37°C) temperatures.
Sporulation time in the environment will depend upon ambient temperature and
sporulated oocysts may be found distant from the pollution source in the aquatic
environment. Sources of pollution with unsporulated oocysts are likely to be
effluent discharges from sewage treatment and waste stabilisation ponds with
detention times of less than 1 week.
Like C. parvum oocysts and G. intestinalis cysts, oocysts of Cyclospora sp. are
likely to survive longer at lower temperatures when suspended in water.
Cyclospora sp. oocysts stored 4°C do not appear to sporulate (Smith et al.,
1997). A proportion of oocysts stored at 4°C for up to 2 months will sporulate
when subsequently incubated at temperatures between 22°C and 37°C. No data
are available regarding survival and transport in soil.
Adequate treatment
Filtration
The principal barrier for these resistant protozoa is physical removal by filtration.
The smaller size of Cryptosporidium oocysts makes them more difficult to
remove than Giardia cysts. Rapid sand filtration is a common treatment process
used to remove particles and when operating efficiently is theoretically capable
of 3 log removal of Cryptosporidium oocysts (Ives, 1990). Other investigations
34
Protozoan parasites
have given a range of removal rates including 91% (Rose et al., 1986) and
greater than 99.999% (Hall et al., 1994b) with the higher removal rates being
achievable when coagulant dosing has been applied to the water prior to
filtration.
Diatomaceous earth filtration has been reported to achieve >99% removal of
Giardia (Jakubowski, 1990) and even up to 4-6 log-units for Cryptosporidium
under laboratory conditions (Ongerth & Hutton, 1997).
Conventional treatment (coagulation, sedimentation, filtration), direct filtration
(with chemical pretreatment) and high-rate filtration can remove 99% of the
(oo)cysts, when properly designed and operated (LeChevallier et al., 1991;
Nieminski, 1994; West et al., 1994). Typically the chemicals used are ferric or
aluminium salts and there appears to be no real difference in the effectiveness
of aluminium sulphate, polyaluminium chloride, ferric sulphate and ferric chloride
in removing oocysts and similarly sized particles (Ives, 1990).
If filters are backwashed, the backwash water may contain high levels of
(oo)cysts (Richardson et al., 1991). If this backwash water is recycled,
treatment with coagulation and sedimentation or microfiltration will reduce recontamination of the water with (oo)cysts. If this is not feasible, it is
recommended that the recycled water is returned at a constant, low rate (Rose
et al., 1997).
Slow sand filtration can efficiently remove (oo)cysts, but the efficiency reduces
at lower temperatures. No data are available for removal of oocysts in full scale
plants but a number of pilot scale studies have been completed where the
removal efficiencies were generally good. Hall et al. (1994) demonstrated
removals of greater than 99.95%. In another study using surface water, heatinactivated oocysts were added at a concentration of 4000 per litre and no
oocysts were found in the filtrate. At the end of the study, intact oocysts were
found only in the upper 2.5 cm of the sand filter (Timms et al., 1995).
Micro- and ultrafiltration can remove over 99.99% (Jacangelo et al., 1991;
Adham et al., 1994; Drozd & Schwartzbrod, 1997) as long as the integrity of
the system is maintained.
Soil passage
Soil passage, used in bank filtration and infiltration, is probably an effective
physical barrier against (oo)cysts. It’s effectiveness depends on travel time and
distance and composition of the soil (Mawdsley et al., 1996).
Pretreatment reservoirs
Storage in reservoirs with a residence time of 5 months can reduce the (oo)cyst
concentration by 99% (Ketelaars et al., 1995). Experimental evidence suggests
that sedimentation of Cryptosporidium oocysts and Giardia cysts is unlikely to
have a significant effect on their removal from a body of water unless they are
attached to other particles (Medema et al., 1998b).
Disinfection
Disinfection with chlorine has always been an important barrier for waterborne
pathogens. The high resistance of especially Cryptosporidium oocysts against
35
Chapter 1
chlorine disinfection (Korich et al., 1990; Smith et al., 1990; Ransome et al.,
1993) renders this process ineffective for oocyst inactivation in drinking water
treatment. Chlorine dioxide is slightly more effective, but still requires a high CT
product (measure of disinfectant dose: (residual) concentration of disinfectant C
x contact time T) of 78 mg.min l-1 for 90% inactivation of oocysts (Korich et al.,
1990). Giardia is less resistant against chlorine: 99.99% reduction can be
achieved with a CT of 180-530, depending on temperature and pH of the water
(Hibler et al., 1987). Chlorine dioxide gives 99% reduction at CT values of 4.728 (Leahy et al., 1987; Rubin, 1988).
Ozone is the most potent (oo)cysticide: at 20°C, the CT for 99% inactivation of
C. parvum oocysts is 3.5 mg.min.l-1 (Finch et al., 1993a) and for G. intestinalis
cysts 0.6 mg.min.l-1 (Finch et al., 1993b). The effectiveness of ozone reduces at
lower temperatures. Peeters et al.(1989) found that 0.4 mg.l-1 residual ozone for
six minutes was sufficient to kill 10 000 oocysts ml-1 whilst Korich et al. (1990)
demonstrated that 1 mg.l-1 for ten minutes at 25°C would result in a reduction
in viability of 99%. Parker et. al. (1993) found that 3 mg.l-1 for ten minutes was
required to kill all oocysts and similar high figures were quoted by Ransome et
al. (1993). Hence, the CT values required for inactivation of cysts and oocysts
are high. CT values are limited, however, since high CT’s can give rise to
formation of high concentrations of (geno)toxic by-products. Exposure of
Cryptosporidium oocysts to multiple disinfectants has been shown to be more
effective than was to be expected from both disinfectants alone (Finch et al.,
1994; Liyanage et al., 1997) and synergism between environmental stress
during sand filtration has also been observed (Parker et al., 1993). The multiple
stresses that (oo)cysts encounter in the environment and during treatment might
limit the infectivity of (oo)cysts.
Conventional UV systems have a limited effect on Cryptosporidium and Giardia
viability. UV doses of 110-120 mJ/cm2 result in 99% inactivation of C. parvum
oocysts (Ransome et al., 1993), as assayed with in vitro viability methods and
97% of G. intestinalis cysts (Rice & Hoff, 1981). A recent study of Clancy et al.
(1998), using animal infectivity, showed that pulsed and advanced UV are much
more effective against Cryptosporidium; they obtained 99.98% inactivation at
UV-doses as low as 19 mJ/cm2. The results of laboratory disinfection
experiments should be translated with caution to the full scale treatment of
environmental (oo)cysts. In surface water treatment, (oo)cysts may be protected
against the disinfectant because they are attached to colloids. On the other
hand, (oo)cysts that have been exposed to environmental stressors may be
more susceptible to disinfectants (Parker et al., 1993). Moreover, the design
and operation of full-scale treatment systems will, in general, be less optimal for
inactivation than the laboratory setting.
The removal of Cryptosporidium oocysts and Giardia cysts by well designed,
maintained and operated treatment processes are summarised in Table 1.
There is little information available regarding the ability of water treatment
processes to remove or inactivate Cyclospora sp. oocysts. As Cyclospora sp.
oocysts (8-10 µm diameter) are larger than C. parvum oocysts but smaller than
G. intestinalis cysts, it is likely that physical removal will be similar to that
36
Protozoan parasites
obtained with Giardia and Cryptosporidium. In an outbreak in Nepal, filtration
and chlorination did not affect the integrity of the oocysts (Rabold et al., 1994).
Although chlorine residuals remained at acceptable levels (0.3 - 0.8 ppm) and
no coliform indicator bacteria were detected, Cyclospora sp. oocysts were
found in the drinking water supply. Little is known about survival of the oocysts
in different environments or what treatment can effectively inactivate the
oocysts.
Risk assessment to design adequate treatment
One of the key issues in treatment is to determine what level of treatment is
adequate. This requires maximum acceptable concentrations in drinking water.
In analogy to toxic compounds, these could be derived from a maximum
acceptable risk and the dose response relation of these parasites. An infection
risk of 10-4
37
Chapter 1
Table 1. Removal of Cryptosporidium oocysts and Giardia cysts by treatment
processes.
Removal efficiency
(10log-units)
Type of process
Cryptosporidium
Giardia
Most important efficiencydetermining parameters
Disinfection processes
Chlorine
0
0-2
Chloramines
0
0-2
Chlorine dioxide
0
0-2
0-2
1-4
0-4
0 - 4?
dose at 254 nm, turbidity,
solutes, system design
0-1
0-1
filtration rate, recycling of
backwash water
presence of “Schmutzdecke”,
filter depth, filtration rate,
temperature
filtration
rate, filter depth,
pore size, precoat thickness,
filter integrity
system
integrity, membrane
type
coagulant dose, pH,
temperature, installation
design, addition of polymers,
Ozone
UV
dose, contact time,
installation design,
disinfectant-demand,
temperature, pH (esp
chlorine), formation of toxic
by-products, synergism of
multiple disinfectants
Filtration processes
Rapid sand filtration
Slow sand filtration
1.2 ->3.7
1.2 ->3.7
Diatomaceous earth
2-6
2-6
Membrane filtration
2 - >4
2 - >4
Coagulation/filtration
2 - 2.5
2 - 2.5
Soil passage
>2 - >5
>2 - >5
Reservoir storage
0.5 - 2
0.5 - 2
Other processes
soil composition, residence
time, travel distance,
presence of
sediment
residence
time,
reservoir
design, temperature
per year has been suggested as acceptable for pathogens in drinking water
(Regli et al., 1991). The maximum concentrations of viable (oo)cysts in drinking
water to meet this risk level are very low (Rose et al., 1991b; 1997). Current
techniques do not allow an evaluation of compliance with these concentrations.
Therefore, safe-guarding of drinking water with respect to protozoan parasites
should be done by a quantitative description of the protozoa concentrations in
the source water and the removal efficiency of the treatment steps. Surface
38
Protozoan parasites
water utilities and groundwater utilities that may be influenced by surface water
or other sources of contamination should monitor their source water for
protozoa and determine the rate of protozoa removal and inactivation achieved
in the treatment plant, in order to determine if acceptable concentrations of
protozoa in drinking water have been achieved.
Verification of efficiency of parasite removal
For routine monitoring, water quality and process parameters are required to
verify treatment performance. Several parameters have been suggested as a
surrogate for (oo)cyst removal by filtration processes: turbidity, particle counts
(LeChevallier & Norton., 1992; Hall & Croll, 1997), clostridial spores (Payment &
Franco, 1993; Hijnen et al, 1997) or aerobic spores (Nieminski, 1997) and
particulate matter (Anon., 1997). Although turbidity or particle counts of filtered
water depend on both the levels in raw water and filter performance, in general,
a turbidity of 0.1 to 0.5 NTU or counts of particles >3 µm below 50 per ml are
indicative of good quality water. On-line monitoring of turbidity or particle
counts gives direct and continuous information on (individual) filter performance
and are very valuable tools for optimising treatment efficiency for (oo)cyst
removal.
Critical moments in the filter cycle are just after backwash or, in case of slow
sand filtration, scraping of the clogged top-layer from the filterbed. A slow
increase in filtration rate or filtering-to-waste minimises the risk of (oo)cyst
breakthrough.
For disinfection processes, disinfectant dose, contact time, residual disinfectant
concentration after this contact time, pH and temperature are commonly used to
monitor the disinfection performance. The most critical conditions for
disinfection processes are low temperatures and high turbidity of the water that
is to be disinfected.
CONCLUSIONS AND RECOMMENDATIONS
Health risk assessment
The abundance and size of drinking waterborne outbreaks in developed
countries show that transmission of Giardia and Cryptosporidium by drinking
water is a significant risk. In the case of Cryptosporidium, the absence of an
adequate cure for immunocompromised patients increases the problem.
Although the outbreaks receive most attention, low-level transmission of these
protozoa through drinking water is very likely to occur in developed countries
and in developing countries alike (Fraser & Cooke, 1991; Isaac-Renton et al.,
1996). Cysts and oocysts are regularly found in drinking water (Isaac-Renton et
al., 1996; Karanis & Seitz, 1996; Rose et al, 1997), although only a small
proportion may be viable and infectious to man. A major drawback for the
determination of the health significance of (oo)cysts in (drinking) water is that
methods for a sensitive and specific detection of infectious (oo)cysts, with a
consistently high recovery are not available.
39
Chapter 1
Risk management
Cryptosporidium poses a serious health risk to immunocompromised persons,
especially AIDS patients. An important step forward in the reduction of the
consequences of waterborne outbreaks and other cases of cryptosporidiosis
would be the definition of an adequate therapy for the immunocompromised
patients. Currently, prevention of exposure to (potentially) contaminated water
is a means to reduce the risk. Especially the immunocompromised population
should be informed about the risk and means to prevent exposure. Boiling of tap
water, use of mineral or bottled water, not swimming in surface water or pools
are some of the options. Local considerations play a major role and public health
authorities are encouraged to provide guidance on the safety of drinking water
for the immunocompromised and on applicable means to reduce exposure
(Anon., 1995a.b; Juranek, 1995). In outbreak situations, rapid investigation of
size and source of the outbreak and installation of control measures to prevent
further transmission is required. Useful guidance on management of waterborne
outbreaks can be found in the report of the UK group of experts (Anon., 1990)
and the CDC Guidance Manual (Juranek, 1995) and a workshop report (Anon.,
1995).
The protozoa, and to a lesser extent the viruses, have changed the philosophy
in the developed countries towards safe-guarding of drinking water from
monitoring of the ‘end-product’ drinking water to monitoring raw water and the
efficiency of the treatment. Furthermore, the extreme resistance of these
organisms implies that a “zero-risk” is no longer achievable. Treatments should
be designed to reduce the (oo)cyst concentrations in the raw water as far as
possible and preferably include filtration step(s). This implies that information on
the parasite concentrations in the raw water is necessary, as well as information
on the removal efficiency of the treatment. Quantitative risk assessment
provides a tool for the combination of information on raw water quality
(concentrations detected, recovery of the detection method, viability) and
treatment efficiency (removal by different steps in the treatment) (Teunis et al.,
1997). The current detection methods are generally sensitive enough to
determine the concentrations of Cryptosporidium and Giardia in surface water,
but are in many cases not sensitive enough for an accurate description of
removal efficiency. For the latter description, data from laboratory studies and
(seeded) pilot plant studies may provide additional information. Another
approach is to determine if an adequate surrogate parameter can be found for
the description of removal efficiency for Cryptosporidium (as Giardia is easier to
eliminate both with disinfection and filtration, the description of treatment
efficiency should be targeted on Cryptosporidium). Several parameters have
been evaluated on a limited scale as surrogates for protozoa removal: aerobic
spores, clostridial spores, particles and algae, but a broader evaluation is
necessary to determine the value of these parameters.
The definition of maximum acceptable concentrations of pathogens in drinking
water based on a maximum acceptable (infection) risk level has become possible
by the availability of volunteer data and dose-response models (Haas, 1983;
Dupont et al., 1995; Teunis et al., 1996).
40
Protozoan parasites
An annual infection risk level of 10-4, as proposed by the US EPA, is currently
used in the US (Rose et al., 1997), Canada (Wallis et al., 1995) and the
Netherlands (Medema et al., 1995) as the basis to determine the appropriate
removal efficiency of surface water treatment systems. Although there are still
questions about the significance of (oo)cyst occurrence in drinking water, this is
the way forward. The implementation of guideline levels is still hampered,
however, by the difficulty to determine source water quality and treatment
efficiency accurately.
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